N-linked glycosylation (NLG) is a co-translational protein modification common to receptor tyrosine kinases (RTKs) as well as most proteins that traverse the secretory pathway. Efficient NLG requires the stepwise biosynthesis of lipid-linked oligosaccharides (LLO), a process initiated in the cytoplasm and completed in the endoplasmic reticulum prior to LLO transfer by oligosaccharyl transferase. We have taken advantage of the sequence specificity of NLG and the sensitivity of the luciferase detection to develop a reporter that measures NLG inhibition in living cells. Following stable expression of this reporter construct in mammalian cells, we optimized a method to perform high throughput screening of compound libraries in 384 well plate formats. In parallel we also developed a second luciferase construct that is insensitive to NLG and thus provides an assay to triage non-specific activators of luciferase. A third screen which identifies characteristic changes in glyco-protein (ie EGFR) localization after disrupting NLG has also been optimized for high content imaging in 96 well plates. In this proposal we set forth a sequential strategy to use these assays to select NLG inhibitors from the MLPCN compound libraries. We also propose additional functional assays performed in medium throughput formats to discriminate between NLG enzymatic steps in order to both confirm compound activity and to identify specific targets and potential mechanisms of action of NLG inhibitors. We provide preliminary data on the feasibility of this screening strategy and predict that the proposed research will identify novel inhibitors of the NLG biosynthetic machinery, providing important tools for the biological sciences as well as potential therapeutics for the treatment of malignant diseases.